We present the first report of enzymatic catalysis in an ionic liquid. The virtually nonexistent vapor pressure makes ionic liquids an exciting new alternative for enzyme-catalyzed syntheses in environmentally friendly environments. Z-aspartame was synthesized in a thermolysin-catalyzed reaction of carbobenzoxy-L-aspartate and L-phenylalanine methyl ester hydrochloride in 1-butyl-3-methylimidazolium hexafluorophosphate (BP6). Ionic liquids such as BP6 are thermally stable and have a remarkable range of temperatures over which they remain liquid (300 degrees C). With an initial rate of 1.2 +/- 0.1 nmol min(-)(1) mg(-)(1), we observed a competitive rate in comparison to that of enzymatic synthesis in organic solvent. Additionally, the enzyme exhibits outstanding stability, which would normally require immobilization.

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We have studied a thermolysin‐catalyzed solid‐to‐solid dipeptide synthesis using equimolar amounts of Z‐Gln‐OH and H‐Leu‐NH2 as model substrates. The high substrate concentrations make this an effective alternative to enzymatic peptide synthesis in organic solvents. Water content was varied in the range of 0 to 600 mL water per mol substrate and enzyme concentration in the range of 0.5 to 10 g/mol of substrates. High yields around 80% conversion and initial rates from 5 to 20 mmol s−1 kg−1 were achieved. The initial rate increases 10‐fold on reducing the water content, to reach a pronounced optimum at 40 mL water per mol substrate. Below this, the rate falls to much lower values in a system with no added water, and to zero in a rigorously dried system. This behavior is discussed in terms of two factors: At higher water contents the system is mass transfer limited (as shown by varying enzyme content), and the diffusion distances required vary. At low water levels, effects reflect the stimulation of the enzymatic activity by water. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:68–72, 1998.

A systematic study of thermolysin‐catalyzed solid‐to‐solid peptide synthesis using Z‐Gln and Leu‐NH2 as model substrates was carried out. The aim was to extend the kinetic knowledge of this new reaction system involving highly concentrated substrate mixtures with little water (10% to 20% w/w). Preheating of the substrates, and ultrasonic treatment, as described in the literature, had no significant effect on our system. The formation of a third compound, the salt of the two substrates, was discovered during melting point experiments. This was associated with a very strong dependence of kinetics on the exact substrate ratio (e.g., twofold higher initial rate with 60% Leu‐NH2 and 40% Z‐Gln than with the equimolar substrate ratio). A model was developed to show how the composition and pH of the liquid phase depends on the substrate ratio, and seemed to explain the experimental rates. In addition, the influences of different mixing and water distribution methods are described. Finally, we can now summarize the major effects of the reaction system as a starting point for further research and scale‐up studies. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 316–321, 1999.

Enzymatic peptide synthesis can be carried out efficiently in solid-to-solid reaction mixtures with 10% (w/w) water added to a mixture of substrates. The final reaction mass contains >/=80% (by weight) of product. This article deals with acid-base effects in such reaction mixtures and the consequences for the enzyme. In the Thermoase-catalyzed synthesis of Z-Asp-Phe-OMe, the reaction rate is strongly dependent on the amount of basic salts added to the system. The rate increases 20 times, as the KHCO(3) or K(2)CO(3) added is raised 2.25-fold from an amount equimolar to the Phe-OMe. HCL starting material. With further increases in KHCO(3) addition, the initial rate remains at the maximum, but with K(2)CO(3) it drops sharply. Addition of NaHCO(3) is less effective, but rates are faster if more water is used. With >1.5 equivalents of basic salt, the final yield of the reaction decreases. Similar effects are observed when thermolysin catalyzes the same reaction, or Z-Gln-Leu-NH(2) synthesis. These effects can be rationalized using a model estimating the pH of these systems, taking into account the possible formation of up to ten different solid phases.